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Single-Cell RT-PCR using RNasin® Ribonuclease Inhibitor

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LabFact #2

RT-PCR should be performed in the absence as well as presence of the reverse transcriptase, to assess DNA contamination in the template RNA. In addition, "no-template" negative control reactions should always be performed.

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Abstract

Experiments were performed to determine whether whole cell lysates made from small cell numbers could be successfully used as direct templates for amplification using the Access RT-PCR System (Cat.# A1250). These experiments were based on a technique by Klebe et al.

Natalie Betz

Promega CorporationPublication Date: 2000

Introduction

Reverse transcription PCR (RT-PCR) is a sensitive and versatile technique for the analysis of gene expression, particularly for rare, low abundance mRNA transcripts. RT-PCR can be performed both qualitatively or quantitatively, and can be useful in cloning novel or homologous RNAs, in differential display, and even as templates for in vitro translation. The RNA template used for RT-PCR is generally purified from the source of interest. This purification process can be both time-consuming and costly, depending on the source of RNA and the method employed. Work by Klebe et al.(1)
suggested that RT-PCR could be performed on whole cell lysates without prior RNA isolation. These researchers developed a technique in which whole cells (generally less than 250) are lysed in a solution containing Recombinant RNasin® Ribonuclease Inhibitor (Cat.# N2511) by freeze/thaw. The whole cell lysate generated is then used as the source of RNA template for RT-PCR.

Experiments were conducted to determine whether RNasin® Ribonuclease Inhibitor could be used in conjunction with the Access RT-PCR System (Cat.# A1250) for successful “single-cell RT-PCR”. The cell type chosen was K562 human erythroleukemia cells and the amplification targets were the bcr-abl transcript and the beta-actin transcript.

Methods and Results

K562 human promyelocytic leukemia cells were diluted in sterile 1X PBS, such that a theoretical given cell number was present in a 5µl volume. The cells were then added to 0.5ml Eppendorf tubes containing 5µl of 2X RNasin® Ribonuclease Inhibitor Freeze Medium (2µl Recombinant RNasin® Ribonuclease Inhibitor [40u/µl] + 18µl 0.15M NaCl/10mM Tris-HCl [pH 8.0]/5mM DTT). The cells were then quickly frozen on dry ice and stored at –70°C. The cells lyse during this freezing, and subsequent thawing, step. The entire 10µl of lysed cells were added directly to 50µl RT-PCR using the Access RT-PCR System, following Technical Bulletin #TB220 (up to 20µl of this freeze media mixture does not interfere with RT-PCR; data not shown). Primers for either the bcr-abl chromosomal translocation specific mRNA found in K562 cells, or β-Actin Primer Pairs (Cat.# G5740), were used (50pmol each per reaction). The following cycling conditions were used: 48°C for 45 minutes, 95°C for 2 minutes, 40 cycles of 94°C for 30 seconds/60°C for 1 minute/68°C for 1 minute, final extension at 68°C for 7 minutes, and soak at 4°C. PCR amplifications were performed using a Perkin Elmer 480 thermocycler.

As seen in Figure 1, the bcr-abl RT-PCR product (200bp) could be detected by agarose gel electrophoresis (1.2%) and ethidium bromide staining from as little as 1 cell, and the signal increased proportionately with cell number (from 1 to 5,000 cells). For the β-actin message (285bp), a signal could be detected in approximately 100–200 cells; as expected, detection increased with increasing cell number (see Figure 1, Panel B). Thus the actual success of this method will be highly dependent on the amplification target, its abundance and how well it can be amplified. The bcr-abl transcript is abundant in K562 cells and the amplification is robust. For more information concerning the bcr-abl transcript in K562 cells, as well as primer sequence, see Tbakhi et al.(2)
.

Conclusion

The combination of RNasin® Ribonuclease Inhibitor and the Access RT-PCR System allowed for the detection of the bcr-abl transcript from as little as a single K562 cell, while the β-actin transcript was detectable from 100-200 K562 cells. The RT-PCR product signal intensity increased with increasing cell number, and for this cell type and targets amplified the technique appears to be able to accommodate up to approximately 5,000 cells. The actual success of this method however, will be highly dependent on the amplification target, its abundance and it amplification efficiency. The bcr-abl transcript is abundant in K562 cells and its amplification is very robust. The work by Klebe et al. used the Animal Injectable Grade of RNasin® Ribonuclease Inhibitor, but these experiments demonstrate that Recombinant RNasin® Ribonuclease Inhibitor is also suitable for single-cell RT-PCR.

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